CN110646402B - A Super-Resolution Fast Scanning Coherent Raman Scattering Imaging Method - Google Patents
A Super-Resolution Fast Scanning Coherent Raman Scattering Imaging Method Download PDFInfo
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Abstract
本发明公开了一种超分辨快速扫描的相干拉曼散射成像方法，其特点是采用DOE与色散元件组成的衍射光学器件组将聚焦到显微物镜上的单束光斑进行不同角度的偏折后为多个1xN光斑入射在生物样品上，激发更小生物分子的反斯托克斯光经聚光、滤波后由探测器进行采集，实现超分辨快速扫描的相干拉曼散射成像。本发明与现有技术相比具有将单束光斑变为多束光斑，减少了行扫描时间，调节两束光的重合面积为切入点，使更小尺寸的生物分子被激发出反斯托克斯光，极大地提高了CARS显微成像的分辨率和振镜扫描效率，使更小尺寸的生物分子被激发出反斯托克斯光，极大提高了振镜扫描效率，实现超高分辨率的CARS显微成像。
The invention discloses a super-resolution fast scanning coherent Raman scattering imaging method. For multiple 1xN light spots incident on the biological sample, the anti-Stokes light that excites smaller biomolecules is collected by the detector after concentrating, filtering, and coherent Raman scattering imaging with super-resolution fast scanning. Compared with the prior art, the invention has the advantages of changing a single beam spot into multiple beam spots, reducing the line scanning time, adjusting the overlapping area of the two beams as the cut-in point, so that the biomolecules of smaller size can be excited out of the anti-Stokes Stokes light, which greatly improves the resolution of CARS microscopic imaging and the scanning efficiency of galvanometers, enables smaller-sized biomolecules to be excited by anti-Stokes light, greatly improves the scanning efficiency of galvanometers, and achieves ultra-high resolution rate of CARS microscopy imaging.
Description
Technical Field
The invention relates to the technical field of spectral imaging, in particular to a coherent Raman scattering imaging method for super-resolution fast scanning.
Background
In recent years, coherent anti-stokes raman scattering microscopy (CARS) research has become mature, which becomes an indispensable imaging mode in biological research by virtue of high resolution and sensitivity, label-free, noninvasive and three-dimensional imaging, thus greatly helping people to acquire biological sample information and leading people to know more abundant microscopic world. As the application of CARS microscopic imaging in biological research becomes more and more widespread, increasing the resolution and imaging rate of CARS microscopic imaging becomes more and more urgent. In CARS microscopic imaging, two beams of pumping light and anti-Stokes light are used, the wavelengths of the two beams of light are changed in an experiment, the frequency difference of the two beams of light is matched with the vibration energy levels of different sample molecules, and the anti-Stokes light is excited. In principle, it is only possible to excite anti-stokes light when the pumping light and stokes light irradiate on the molecule simultaneously, so that the spatially overlapped area of the two beams determines the resolution of the CARS imaging, and theoretically the smaller the overlapped area, the higher the resolution.
In the prior art, coherent Raman scattering imaging adopts progressive scanning, namely light spots are scanned from the leftmost end to the rightmost end of a first line of a sample, the scanning is started by jumping to a second line after the scanning of the first line is finished, and the scanning is repeated in a circulating way, so that the scanning efficiency is low, and the resolution is not high. Since the diffraction limit cannot be broken, it is difficult to distinguish smaller sized biomolecules, which has great limitations in medical and related biological applications.
Disclosure of Invention
The invention aims at the defects of the prior art to design a coherent Raman scattering imaging method of super-resolution fast scanning, which adopts a CARS micro-imaging system of DOE and a dispersion element to change a single light spot into a plurality of light spots on the premise of not changing the physical characteristics of original laser, reduces the line scanning time, can realize the purpose of scanning one line only by deflecting the X axis of a galvanometer by a small angle, greatly shortens the time for scanning one image, greatly improves the resolution of the micro-imaging of the galvanometer and the scanning efficiency of the galvanometer by adjusting the superposition area of the two beams as an entry point, ensures that small-sized biomolecules are excited to generate anti-Stokes light, ensures that the seen image is clearer, greatly improves the resolution of the biological imaging, ensures that the details of a biological sample are richer, realizes fast scanning and ultrahigh resolution, and obviously improves the imaging speed and resolution, the method is simple and convenient, is convenient to operate, has high reliability, widens the application field, and particularly provides great benefits and help for application research and basic research in the field of spectral imaging.
The purpose of the invention is realized as follows: a coherent Raman scattering imaging method of super-resolution fast scanning comprises a CARS microscopic imaging method that pump light and Stokes light are combined and enter a galvanometer after beam expanding/collimating, then the pump light and the Stokes light are focused on a back focal plane of a microscope objective through a scanning lens/tube lens group and are incident on a biological sample, and excited anti-Stokes light is collected by a detector.
The Diffractive Optical Element (DOE) is combined with dispersion elements with different parameters, the area of a superposition area of the pump light and the Stokes light is adjusted to be small enough, and coherent Raman scattering imaging with ultrahigh resolution is realized.
Compared with the prior art, the invention has the advantages that on the premise of not changing the physical characteristics of the original laser, a single light spot is changed into a plurality of light spots, the line scanning time is reduced, the purpose of scanning one line can be realized only by deflecting the X axis of the galvanometer by a small angle, the time for scanning one image is greatly shortened, the overlapping area of two beams of light is adjusted as an entry point, the resolution ratio of CARS microscopic imaging and the scanning efficiency of the galvanometer are greatly improved, small-sized biomolecules are excited to generate anti-Stokes light, the details of a biological sample are richer, the rapid scanning is realized, the ultrahigh resolution ratio is realized, the imaging speed and resolution ratio are obviously improved, the method is simple and convenient, the operation is convenient and fast, the reliability is high, the application field is widened, and particularly great benefits and help are provided for application research and basic research in the field of spectral imaging.
Drawings
FIG. 1 is a schematic diagram illustrating the number variation of light spots after passing through a DOE;
FIG. 2 is a schematic diagram showing the change of the overlapping area of the light spot before and after the light spot passes through the DOE and the dispersion element;
FIG. 3 is a schematic diagram showing changes in laser intensity distribution before and after a light spot passes through a DOE;
FIG. 4 is a schematic representation of CARS microscopic imaging;
FIG. 5 is a diagram of an embodiment of forward probe coherent Raman scattering imaging;
FIG. 6 is a diagram of an embodiment of back-probed coherent Raman scattering imaging.
Detailed Description
The invention adopts the diffractive optical device group formed by combining the DOE and the dispersion element to change the single-beam light spot into the multi-beam light spot, wherein the dispersion elements with different parameters and the DOE act together to flexibly adjust the overlapping area of the pump light and the Stokes light, so that the overlapping area is small enough to realize the purpose of super-resolution imaging. The DOE divides the beam of a single light spot to obtain a plurality of (1xN) light spots, and the scanning speed is improved. The invention includes laser sources of 1030nm and 790 nm; a positive and negative lens group which expands and collimates the beam; a lens (i.e., an achromatic lens) for eliminating chromatic aberration of the pump light and the stokes light; selecting a dichroic mirror which reflects 790nm pump light and 1030nm Stokes light and transmits 640nm anti-Stokes light; a scanning lens and a tube lens group which ensure that the light beam coming out of the galvanometer does not deviate from the microscope objective and is focused on the back focal plane of the tube lens; scanning a galvanometer; a filter plate; and the CARS microscopic imaging method comprises a PMT detector, a data acquisition card for recording and storing acquired data and the like which form an acquisition light path.
The pump light and the Stokes light are simultaneously irradiated on the biomolecules to possibly excite the anti-Stokes light, and only the superposed region of the two beams of light is an effective region. The invention can flexibly change the area of the overlapping area of the light spots of the two beams of light to be smaller by deflecting the pumping light and the Stokes light at different angles through the dual functions of the DOE and the optical dispersion element, thereby distinguishing smaller biomolecules. The diffraction optical device group combined by the DOE and the optical dispersion element changes the light intensity distribution of the two laser beams of the pumping light and the Stokes light from the original Gaussian distribution into the current sharp edge, so that the energy is concentrated on the overlapped part of the two laser beams, and the anti-Stokes light is easier to excite. And after the DOE is adopted, on the premise of not changing the properties of the two beams of light, the single beam of light spot is changed into the multiple beams of light spots, the line scanning time is greatly reduced, the X axis of the galvanometer only needs to deflect a small angle to realize the purpose of scanning one line, and the time for scanning one image is greatly shortened.
The present invention is further described in detail below with reference to changes in light spot and light intensity of pump light and stokes light after passing through a Diffractive Optical Element (DOE), and the DOE controlling the phase of incident light by using a micro-nano structure on a lens surface to realize modulation of laser light, such as laser beam splitting, laser sampling, and laser shaping.
Referring to the attached drawing 1, after pump light and stokes light are combined/collimated and achromatic, a single light beam entering one light spot 18 of a scanning lens/tube lens group through a galvanometer is split by a Diffractive Optical Element (DOE)8 to become a plurality of light beams of a plurality of light spots 18, the split light beams are arranged according to a one-dimensional shape (1xN), the diameter and the phase of each light beam are consistent with those of an incident light beam, the diffraction efficiency is 70-95%, the difference of the diameter energy of the light beam is less than 1%, and the spacing distance between adjacent light spots is calculated by the following formula (a):
L＝d*tan(Φ) (a)
in the formula: d is the vertical distance from the laser to the surface of the sample; phi is the angle between adjacent laser beams.
Referring to fig. 2a, when the pump light emitted by the 790nm laser 1-1 and the stokes light emitted by the 1030nm laser 1-2 pass through the single beam of light emitted by the beam combiner 2, diffraction angles of the light with different wavelengths in the DOE are different, and then the two beams of laser light both have deflection with different angles after exiting the DOE, because the wavelength difference of the two beams of laser light is large, the situation that the light spots 18 of the two beams of laser light are completely separated may occur, that is, the area of the overlapping area of the two light spots 18 is zero, and the condition of coherent raman imaging cannot be satisfied at this time.
Referring to fig. 2b, by placing the dispersion element 9 after the Diffractive Optical Element (DOE)8, it also appears that the area of the overlapping region 19 of the spots 18 of the two laser beams becomes large. In practical applications, the DOE is not flexible enough to deflect the pumping light and the stokes light, and the coherent raman imaging may be affected by complete separation of the spots 18 of the two beams or an excessively large area of the overlapping region 19.
Referring to fig. 2c, a dispersion element 9 with suitable parameters is arranged behind a Diffractive Optical Element (DOE)8, so that the area of a region 19 where the pump light and the stokes light overlap 18 can be flexibly adjusted. The superposed part of the two beams of light is finely adjusted to be small enough to break through the diffraction limit so as to achieve the purpose of super-resolution imaging. To meet the experimental requirements, the refractive index of the Diffractive Optical Element (DOE)8 is generally n1, and the refractive index of the dispersive element 9 is n 2.
Referring to fig. 3a, the intensity of the laser light before passing through the DOE is gaussian.
Referring to fig. 3b, the light intensity of the laser passing through the DOE has a steep edge distribution, so that the light intensity is concentrated on the overlapped part of the light spots, and the light intensity is ensured to be enough to excite the anti-stokes light.
Referring to fig. 4, the present invention is based on the four-wave mixing principle, different from spontaneous raman and other raman scattering effects, and matches the frequency difference between stokes light and pump light with the molecular raman mode to be excited, so as to excite anti-stokes light, in which: wpIs pump light; wsIs stokes light; wasIs anti-stokes light; omegaRIs the difference in energy levels of the excited molecules. By the formula: omegaP-ωS＝ωAS-ωPIt can be seen that by adjusting the frequency difference between the stokes light and the pump light, different raman modes can be selectively excited, with great flexibility.
The following describes the present invention in further detail in terms of specific embodiments and procedures for coherent raman scattering imaging.
Example 1
Referring to the attached figure 5, a forward detection coherent raman scattering imaging system of a dual laser 1 is composed of a 1580nm erbium-doped laser 1-1 and a 1030nm ytterbium-doped laser 1-2, the 1580nm erbium-doped laser 1-1 generates 790nm pulse laser serving as pump light through certain nonlinear action, 1030nm pulse light generated by the 1030nm ytterbium-doped laser 1-2 serves as stokes light, and two beams of light form a beam after being combined by a beam combiner 2. Because the laser has certain divergence and the optical path of the coherent Raman scattering imaging system is longer, even if the divergence angle of the laser is smaller, the spot area of the laser transmitted to the entrance pupil of the objective lens 10 is likely to be enlarged, and the invention adopts the lens group 3 consisting of the negative lens 3-1 and the positive lens 3-2 to expand and collimate the beam. The focal length of the negative lens 3-1 is 5mm, the focal length of the positive lens 3-2 is 10mm, the beam expansion multiple of the lens group 3 is f2/f1 which is 2 times, the diameter of a light spot after beam expansion is changed from 2mm to about 4mm, the area of the light spot is smaller than the mirror surface of the galvanometer 6, and the entrance pupil of the objective lens 10 can be filled just. Because light with different wavelengths has different chromatic dispersion and refractive index when passing through the lens group 3, the light with different wavelengths has different focuses, the achromatic lens 4 is adopted for compensation, the pump light and the Stokes light with reduced chromatic dispersion are incident on the X mirror surface of the galvanometer 6 through the reflector 5, and are incident on the Y mirror surface through the reflection of the X mirror surface, and finally are emitted from the galvanometer 6. Since the galvanometer 6 is in a state of being constantly moved during scanning, the light beam emitted from the galvanometer 6 is also in a state of being constantly deflected, and thus the light beam may deviate from the entrance pupil of the objective lens 10, and may deviate from the entrance pupil of the objective lens 10 due to divergence after passing through a long optical path. The invention adopts the scanning lens/tube lens group 7 to refocus the light beam to the back focal plane of the objective lens 10, and the scanning lens/tube lens group 7 arranged behind the galvanometer 6 ensures that the galvanometer 6 can still scan the light beam through the entrance pupil of the objective lens 10 at a larger angle. The scanning plane is a plane emitted from the galvanometer 6, the scanning plane is positioned at the front focal point of the scanning lens 7-1, the rear focal point of the scanning lens 7-1 and the front focal point of the tube lens 7-2 are positioned at the same position, and even if the deflection angle of the galvanometer 6 is larger, a fixed focal point can be formed on the rear focal plane of the tube lens 7-2. Through the continuous scanning of the galvanometer 6, the 1xN light spots are rapidly scanned on the biological sample line by line, and the X mirror surface of the galvanometer 6 can be scanned in one line only by deflecting a small angle, so that the aim of rapid scanning is really fulfilled compared with the single light spot in the prior art. Meanwhile, the smaller coincidence area of the pump light and the Stokes light can excite anti-Stokes light on smaller biomolecules, so that the smaller biomolecules can be distinguished, and the purpose of super-resolution imaging is realized.
The pump light and the stokes light focused by the scanning lens/tube lens group 7 pass through the beam splitting and shaping of a Diffraction Optical Element (DOE)8 and a dispersion element 9, are incident on the biological sample on the objective table 15 through an objective lens 10, and the CARS signal excited by the biological sample enters a filter 12 through a condenser 11. The filter 12 is a band-pass filter, the central wavelength of the filter is 650nm, the bandwidth of the filter is 40nm, and other light wave signals are filtered out and then collected by a PMT detector 13; the PMT detector 13 processes the acquired CARS signals through a computer to obtain a CARS image, and the CARS image is recorded and stored by the data acquisition card 14.
Example 2
Referring to fig. 6, a back detection coherent raman imaging system composed of a 1030nm single laser 1, a photonic crystal fiber oscillator (PCF)2, a first filter 3, a negative lens 4, a positive lens 5, an achromatic lens 6, a galvanometer 7, a scanning lens/tube lens group 8, a Diffractive Optical Element (DOE)11, a dispersive element 12, an objective lens 13, a dichroic mirror 14, a second filter 15 and a detector 16, wherein a diffractive optical device group composed of the Diffractive Optical Element (DOE)11 and the dispersive element 12 makes light beams focused on the objective lens 13 incident on a biological sample after being deflected at different angles, and excites anti-stokes light of smaller biomolecules to be collected by the detector 16 after passing through the dichroic mirror 14 and the second filter 15, and is recorded and stored by a data acquisition card 17; the Diffractive Optical Element (DOE)11 is combined with the dispersion element 12 with different parameters, so that the overlapping area of two light spots of the pump light and the stokes light is small enough, and coherent raman scattering imaging with ultrahigh resolution is realized.
1030nm light generated by the 1030nm single laser 1 is introduced into a Photonic Crystal Fiber (PCF)2, four-wave mixing action is generated in the PCF, generated 790nm pump light, 1479nm idler frequency light and Stokes light are subjected to beam expansion and collimation through a negative lens 4, a positive lens 5 and an achromatic lens 6 after 1479nm idler frequency light is filtered out through a first filter plate 3, and then are incident on an X mirror surface of a vibrating mirror 7, and are reflected on a Y mirror surface through the X mirror surface, laser emitted from the vibrating mirror 7 is focused through a scanning lens/tube lens group 8, and the focused pump light and the focused Stokes light enter a Diffraction Optical Element (DOE)11 and a dispersion element 12 through a first reflecting mirror 9 and a second reflecting mirror 10.
The focal length of the negative lens 4 is 5mm, the focal length of the positive lens 5 is 10mm, the beam expansion multiple of the negative lens is f2/f1 which is 2 times, the diameter of a light spot after beam expansion is changed from the original 2mm to about 4mm, the area of the light spot is smaller than the mirror surface of the galvanometer 7, and the entrance pupil of the objective lens 13 can be just filled. Because the light with different wavelengths has different dispersion and refraction coefficients when passing through the positive/negative lens group, the light with different wavelengths has different focuses, and is compensated by the achromatic lens 6, the pump light and the Stokes light which reduce the dispersion are incident on the X mirror surface of the galvanometer 7, and are reflected by the X mirror surface to be incident on the Y mirror surface and finally emitted from the galvanometer 7. During scanning, the galvanometer 7 is in a continuous motion state, so that the light beam emitted from the galvanometer 7 is in a continuous deflection state, the light beam deviates from the entrance pupil of the objective lens 13, and the light beam also diverges and deviates from the entrance pupil of the objective lens 13 after passing through a long optical path. The scanning lens/tube lens group 8 is adopted to refocus the light beam to the back focal plane of the objective lens 13, and the scanning lens/tube lens group 8 arranged behind the galvanometer 7 ensures that the galvanometer 7 can still scan the light beam through the entrance pupil of the objective lens 13 at a larger angle. The scanning plane emergent from the galvanometer 7 is positioned at the front focus of the scanning lens 8-1, the back focus of the scanning lens 8-1 and the front focus of the tube lens 8-2 are positioned at the same position, and even if the deflection angle of the galvanometer 7 is larger, a fixed focus can be formed on the back focal plane of the tube lens 8-2. The pump light and stokes light focused by the scanning lens/tube lens group 8 are split and shaped by the Diffraction Optical Element (DOE)11 and the dispersion element 12, and finally enter the biological sample on the objective table, and the excited small biological molecule CARS signal enters the second filter 15 through the dichroic mirror 14. The second filter 15 is a band-pass filter, the central wavelength of the second filter is 650nm, the bandwidth of the second filter is 40nm, and other light wave signals are collected by the detector 15 after being filtered; the detector 15 processes the collected CARS signals through a computer to obtain a CARS image, and the CARS image is recorded and stored by a data acquisition card 17. Through the continuous scanning of the galvanometer 7, the 1xN light spots are rapidly scanned on the biological sample line by line, and the X mirror surface of the galvanometer 7 can be scanned in one line only by deflecting a small angle, so that the aim of rapid scanning is really fulfilled compared with the single light spot in the prior art. Meanwhile, the Diffractive Optical Element (DOE)11 is combined with the dispersion element 12 with different parameters, so that the overlapping area of the two light spots of the pump light and the stokes light is small enough, anti-stokes light can be excited on smaller biomolecules, the smaller biomolecules can be resolved, and the purpose of super-resolution imaging is achieved.
After the pump light and the Stokes light pass through a Diffraction Optical Element (DOE)11 and a dispersion element 12, laser beam splitting and ideal light spot overlapping areas are realized; the boundary wavelength of the dichroic mirror 14 is 655nm, when the light beam and the dichroic mirror 14 form an angle of 45 degrees, if the wavelength is more than 668nm, the reflectivity of the light reaches 98 percent, and the light is almost totally reflected and belongs to a reflection area; if the wavelength is less than 642nm, the transmittance of light reaches 96%, and the light belongs to the transmission region. The pump light and the Stokes light are reflected by the dichroic mirror 14, pass through the objective lens 13, and finally irradiate on a sample, the excited 640nm anti-Stokes light can penetrate through the dichroic mirror 14, is filtered by the second filter 15 and then is collected by the detector 16, signals of the light are sent to the data acquisition card 17 for storage, and the collected signals are processed by the computer to obtain a CARS image.
The invention has been described in further detail in order to avoid limiting the scope of the invention, and it is intended that all such equivalent embodiments be included within the scope of the following claims.
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